Approaches to cell based therapies for type 1 diabetes have largely focused on the expression of a single or a combination of transcription factors that can drive ( cell specific patterns of gene expression, and most have relied at least in part on Pdx1, a homeodomain transcription factor with critical regulatory roles in early pancreas development and in the mature ( cell, and on Ngn3, a critical pro-endocrine transcription factor whose transient expression characterizes endocrine progenitors. However promising, the efficiency of achieving a ( cell phenotype by any of these approaches remains low, and the fidelity and stability of these efforts have not been fully evaluated. Although endoderm is achieved reproducibly and efficiently, subsequent transitions, such as from pancreatic progenitor to endocrine pancreatic progenitor, are achieved with particularly low efficiency. We recently determined that Pdx1 contributes to specification of endocrine progenitors in mice both by regulating Ngn3 directly in concert with Hnf6 and by participating in a cross-regulatory transcription factor network during early pancreas development. We hypothesize that understanding the epigenetic regulation of the transcription factor Ngn3 and the roles of Pdx1 and Hnf6 in the cross-regulatory transcription factor network of developing ( cells will inform efforts to improve the efficiency, fidelity and stability of the ( cell phenotype achieved through the guided differentiation of non-( cell populations. Therefore, we propose in AIM 1: To examine the genetic and epigenetic regulation of Ngn3 in human pancreas progenitors, in AIM 2: To analyze the genetic and molecular interactions between Pdx1 and Hnf6 in promoting endocrine progenitor specification, and in AIM 3: To define the role of Pdx1 and Hnf6 in the transcriptional network of endocrine pancreas progenitors through global occupancy and gene expression analysis. The overarching long-term goal is to utilize this information gained from these studies to optimize the guided transition of ES and iPS cells, and possibly mature lineages, toward a ( cell fate. PUBLIC HEALTH RELEVANCE: The high hopes for cell based therapy for type 1 diabetes have been tempered by the limited availability and short-term function of human islets for transplantation, leading to intense focus on alternate sources of (?cells. The overarching goal of the proposed studies is to optimize the guided transitions of ES and iPS cells toward a ( cell fate to make potentially patient-specific ( cells available for transplantation.